Identification Of Interaction With PIP2;4 And Preliminary Functional Analysis Of Cucumber PIP1;2

Cucumber（Cucumis sativus L.）is one of the major vegetable crops cultivated in facilities in China,which requires large amounts of water during its reproductive period and is particularly sensitive to water deficit,and is susceptible to drought stress,leading to yield and quality loss.PIPs are divided into two subclasses,PIP1 and PIP2,and PIP2 has stronger water transport capacity than PIP1,but PIP1 can improve its water transport capacity by interacting with PIP2.It has been found in the literature and in the previous studies of our group that CsPIP1;2 and CsPIP2;4 are highly responsive proteins to drought stress in cucumber,and there may be interactions,and overexpression of CsPIP2;4 significantly increases drought tolerance.In view of this,the present study was conducted to identify the interactions between CsPIP1;2 and CsPIP2;4,and to perform the preliminary analysis of CsPIP1;2 function.The main findings are as follows.1.Bioinformatics analysis revealed that the CsPIP1;2 is relatively evolutionarily conserved,with high functional homology from similar proteins of other species in the same genus;with an average hydrophobicity of 0.338,it is a hydrophilic protein;CsPIP1;2 has six transmembrane structural domains conserved by members of the AQP family,and its NPA structural domain,ar/R selectivity filter and Forger’s residues conserved structural domains are consistent with other homologous proteins;subcellular localization predict its localization to the cytoplasmic membrane;promoter cis-acting elements predict that CsPIP1;2 is involved in the response to adversity and is closely related to the response to adversity involved in ABA;RNA-seq data analysis reveals that CsPIP1;2 is expressed in all tissues of cucumber.2.Normal growth and color turn blue of yeast cells on four-deficient medium were observed in DUAL membrane yeast two-hybrid system,indicating the existence of CsPIP1;2 and CsPIP2;4interactions;bimolecular fluorescence complementation（BiFC）detected a significant fluorescence signal on the cell membrane of co-transformed tobacco,further confirming that CsPIP1;2 and CsPIP2;4 in plant interactions existed in vivo.3.Overexpressed CsPIP1;2 transgenic cucumber was obtained.Compared with wild-type cucumber,the overexpressed CsPIP1;2 cucumber seeds germinated faster,had longer hypocotyls,smaller node spacing and leaves,longer root systems and more lateral roots;the transgenic cucumber leaves showed stronger water retention capacity than wild-type cucumber;under drought stress,the CsPIP1;2 transgenic cucumber showed less change in chlorophyll content compared with wild-type cucumber and had lower peroxidase（POD）and ascorbic acid levels.（POD）and ascorbate peroxidase（APX）activities as well as malondialdehyde（MDA）,soluble protein and hydrogen peroxide（H₂O₂）contents,indicating that CsPIP1;2 transgenic cucumber was less stressed by drought.The above indicates that overexpression of CsPIP1;2 transgenic cucumber has stronger drought tolerance.